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Environmental and Experimental Botany 61 (2007) 199223

Review

Heat tolerance in plants: An overview


A. Wahid a , S. Gelani a , M. Ashraf a , M.R. Foolad b,
a

Department of Botany, University of Agriculture, Faisalabad 38040, Pakistan


Department of Horticulture, The Pennsylvania State University, University Park, PA 16802, USA
Received 30 October 2006; received in revised form 16 May 2007; accepted 27 May 2007

Abstract
Heat stress due to increased temperature is an agricultural problem in many areas in the world. Transitory or constantly high temperatures
cause an array of morpho-anatomical, physiological and biochemical changes in plants, which affect plant growth and development and may
lead to a drastic reduction in economic yield. The adverse effects of heat stress can be mitigated by developing crop plants with improved
thermotolerance using various genetic approaches. For this purpose, however, a thorough understanding of physiological responses of plants to
high temperature, mechanisms of heat tolerance and possible strategies for improving crop thermotolerance is imperative. Heat stress affects
plant growth throughout its ontogeny, though heat-threshold level varies considerably at different developmental stages. For instance, during
seed germination, high temperature may slow down or totally inhibit germination, depending on plant species and the intensity of the stress. At
later stages, high temperature may adversely affect photosynthesis, respiration, water relations and membrane stability, and also modulate levels
of hormones and primary and secondary metabolites. Furthermore, throughout plant ontogeny, enhanced expression of a variety of heat shock
proteins, other stress-related proteins, and production of reactive oxygen species (ROS) constitute major plant responses to heat stress. In order
to cope with heat stress, plants implement various mechanisms, including maintenance of membrane stability, scavenging of ROS, production of
antioxidants, accumulation and adjustment of compatible solutes, induction of mitogen-activated protein kinase (MAPK) and calcium-dependent
protein kinase (CDPK) cascades, and, most importantly, chaperone signaling and transcriptional activation. All these mechanisms, which are
regulated at the molecular level, enable plants to thrive under heat stress. Based on a complete understanding of such mechanisms, potential genetic
strategies to improve plant heat-stress tolerance include traditional and contemporary molecular breeding protocols and transgenic approaches.
While there are a few examples of plants with improved heat tolerance through the use of traditional breeding protocols, the success of genetic
transformation approach has been thus far limited. The latter is due to limited knowledge and availability of genes with known effects on plant
heat-stress tolerance, though these may not be insurmountable in future. In addition to genetic approaches, crop heat tolerance can be enhanced
by preconditioning of plants under different environmental stresses or exogenous application of osmoprotectants such as glycinebetaine and
proline. Acquiring thermotolerance is an active process by which considerable amounts of plant resources are diverted to structural and functional
maintenance to escape damages caused by heat stress. Although biochemical and molecular aspects of thermotolerance in plants are relatively
well understood, further studies focused on phenotypic flexibility and assimilate partitioning under heat stress and factors modulating crop heat
tolerance are imperative. Such studies combined with genetic approaches to identify and map genes (or QTLs) conferring thermotolerance will
not only facilitate marker-assisted breeding for heat tolerance but also pave the way for cloning and characterization of underlying genetic factors
which could be useful for engineering plants with improved heat tolerance.
2007 Published by Elsevier B.V.
Keywords: Heat stress; Heat tolerance; High temperature; Thermotolerance; Stress response; Heat shock proteins; Tolerance mechanism; Molecular breeding;
Transgenic plants

Contents
1.
2.
3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Heat-stress threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plant responses to heat stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Corresponding author. Tel.: +1 814 865 5408; fax: +1 814 863 6139.
E-mail address: mrf5@psu.edu (M.R. Foolad).

0098-8472/$ see front matter 2007 Published by Elsevier B.V.


doi:10.1016/j.envexpbot.2007.05.011

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3.1.

Morpho-anatomical and phenological responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


3.1.1. Morphological symptoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2. Anatomical changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.3. Phenological changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Physiological responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1. Waters relations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2. Accumulation of compatible osmolytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.3. Photosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.4. Assimilate partitioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.5. Cell membrane thermostability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.6. Hormonal changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.7. Secondary metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Molecular responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.1. Oxidative stress and antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.2. Stress proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Mechanism of heat tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Acquired thermotolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Temperature sensing and signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7. Genetic improvement for heat-stress tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1. Conventional breeding strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2. Molecular and biotechnological strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8. Induction of heat tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9. Energy economics under heat stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10. Conclusion and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
Heat stress is often defined as the rise in temperature beyond a
threshold level for a period of time sufficient to cause irreversible
damage to plant growth and development. In general, a transient
elevation in temperature, usually 1015 C above ambient, is
considered heat shock or heat stress. However, heat stress is a
complex function of intensity (temperature in degrees), duration,
and rate of increase in temperature. The extent to which it occurs
in specific climatic zones depends on the probability and period
of high temperatures occurring during the day and/or the night.
Heat tolerance is generally defined as the ability of the plant
to grow and produce economic yield under high temperatures.
However, while some researchers believe that night temperatures
are major limiting factors others have argued that day and night
temperatures do not affect the plant independently and that the
diurnal mean temperature is a better predictor of plant response
to high temperature with day temperature having a secondary
role (Peet and Willits, 1998).
Heat stress due to high ambient temperatures is a serious
threat to crop production worldwide (Hall, 2001). Gaseous
emissions due to human activities are substantially adding to
the existing concentrations of greenhouse gases, particularly
CO2 , methane, chlorofluorocarbons and nitrous oxides. Different global circulation models predict that greenhouse gases
will gradually increase worlds average ambient temperature.
According to a report of the Intergovernmental Panel on Climatic Change (IPCC), global mean temperature will rise 0.3 C
per decade (Jones et al., 1999) reaching to approximately 1 and
3 C above the present value by years 2025 and 2100, respectively, and leading to global warming. Rising temperatures may
lead to altered geographical distribution and growing season of

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agricultural crops by allowing the threshold temperature for the


start of the season and crop maturity to reach earlier (Porter,
2005).
At very high temperatures, severe cellular injury and even
cell death may occur within minutes, which could be attributed
to a catastrophic collapse of cellular organization (Schoffl et
al., 1999). At moderately high temperatures, injuries or death
may occur only after long-term exposure. Direct injuries due
to high temperatures include protein denaturation and aggregation, and increased fluidity of membrane lipids. Indirect or
slower heat injuries include inactivation of enzymes in chloroplast and mitochondria, inhibition of protein synthesis, protein
degradation and loss of membrane integrity (Howarth, 2005).
Heat stress also affects the organization of microtubules by splitting and/or elongation of spindles, formation of microtubule
asters in mitotic cells, and elongation of phragmoplast microtubules (Smertenko et al., 1997). These injuries eventually lead
to starvation, inhibition of growth, reduced ion flux, production
of toxic compounds and reactive oxygen species (ROS) (Schoffl
et al., 1999; Howarth, 2005).
Immediately after exposure to high temperatures and perception of signals, changes occur at the molecular level altering the
expression of genes and accumulation of transcripts, thereby
leading to the synthesis of stress-related proteins as a stresstolerance strategy (Iba, 2002). Expression of heat shock proteins
(HSPs) is known to be an important adaptive strategy in this
regard (Feder and Hoffman, 1999). The HSPs, ranging in
molecular mass from about 10 to 200 kDa, have chaperonelike functions and are involved in signal transduction during
heat stress (Schoffl et al., 1999). The tolerance conferred by
HSPs results in improved physiological phenomena such as
photosynthesis, assimilate partitioning, water and nutrient use

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Table 1
Threshold high temperatures for some crop plants
Crop plants

Threshold temperature ( C)

Growth stage

References

Wheat
Corn
Cotton
Pearl millet
Tomato
Brassica
Cool season pulses
Groundnut
Cowpea
Rice

26
38
45
35
30
29
25
34
41
34

Post-anthesis
Grain filling
Reproductive
Seedling
Emergence
Flowering
Flowering
Pollen production
Flowering
Grain yield

Stone and Nicolas (1994)


Thompson (1986)
Rehman et al. (2004)
Ashraf and Hafeez (2004)
Camejo et al. (2005)
Morrison and Stewart (2002)
Siddique et al. (1999)
Vara Prasad et al. (2000)
Patel and Hall (1990)
Morita et al. (2004)

efficiency, and membrane stability (Camejo et al., 2005; Ahn


and Zimmerman, 2006; Momcilovic and Ristic, 2007). Such
improvements make plant growth and development possible
under heat stress. However, not all plant species or genotypes
within species have similar capabilities in coping with the heat
stress. There exists tremendous variation within and between
species, providing opportunities to improve crop heat-stress
tolerance through genetic means. Some attempts to develop
heat-tolerant genotypes via conventional plant breeding protocols have been successful (Ehlers and Hall, 1998; Camejo et
al., 2005). Recently, however, advanced techniques of molecular breeding and genetic engineering have provided additional
tools, which could be employed to develop crops with improved
heat tolerance and to combat this universal environmental adversary. However, to assure achievement of success in this strategy,
concerted efforts of plant physiologist, molecular biologists and
crop breeders are imperative.
This review accentuates on plant responses and adaptations
to heat stress at the whole plant, cellular and sub-cellular levels,
tolerance mechanisms and strategies for genetic improvement
of crops with heat-stress tolerance.
2. Heat-stress threshold
A threshold temperature refers to a value of daily mean
temperature at which a detectable reduction in growth begins.
Upper and lower developmental threshold temperatures have
been determined for many plant species through controlled laboratory and field experiments. A lower developmental threshold
or a base temperature is one below which plant growth and
development stop. Similarly, an upper developmental threshold is the temperature above which growth and development
cease. Knowledge of lower threshold temperatures is important in physiological research as well as for crop production.
Base threshold temperatures vary with plant species, but for
cool season crops 0 C is often the best-predicted base temperature (Miller et al., 2001). Cool season and temperate crops
often have lower threshold temperature values compared to
tropical crops. Upper threshold temperatures also differ for different plant species and genotypes within species. However,
determining a consistent upper threshold temperature is difficult because the plant behavior may differ depending on other
environmental conditions (Miller et al., 2001). In tomato, for

example, when the ambient temperature exceeds 35 C, its seed


germination, seedling and vegetative growth, flowering and fruit
set, and fruit ripening are adversely affected. For other plant
species, the higher threshold temperature may be lower or higher
than 35 C. Upper threshold temperatures for some major crop
species are diplayed in Table 1. High temperature sensitivity
is particularly important in tropical and subtropical climates as
heat stress may become a major limiting factor for field crop
production.
Brief exposure of plants to high temperatures during seed
filling can accelerate senescence, diminish seed set and seed
weight, and reduce yield (Siddique et al., 1999). This is because
under such conditions plants tend to divert resources to cope
with the heat stress and thus limited photosynthates would be
available for reproductive development. Another effect of heat
stress in many plant species is induced sterility when heat is
imposed immediately before or during anthesis. Pulse legumes
are particularly sensitive to heat stress at the bloom stage; only a
few days of exposure to high temperatures (3035 C) can cause
heavy yield losses through flower drop or pod abortion (Siddique
et al., 1999). In general, base and upper threshold temperatures
vary in plant species belonging to different habitats. Thus, it
is highly desirable to appraise threshold temperatures of new
cultivars to prevent damages by unfavorable temperatures during
the plant ontogeny.
3. Plant responses to heat stress
3.1. Morpho-anatomical and phenological responses
3.1.1. Morphological symptoms
In tropical climates, excess of radiation and high temperatures are often the most limiting factors affecting plant growth
and final crop yield. High temperatures can cause considerable pre- and post-harvest damages, including scorching of
leaves and twigs, sunburns on leaves, branches and stems, leaf
senescence and abscission, shoot and root growth inhibition,
fruit discoloration and damage, and reduced yield (Guilioni et
al., 1997; Ismail and Hall, 1999; Vollenweider and GunthardtGoerg, 2005). Similarly, in temperate regions, heat stress has
been reported as one of the most important causes of reduction in yield and dry matter production in many crops, including
maize (Giaveno and Ferrero, 2003).

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High-temperature-induced modifications in plants may be


direct as on existing physiological processes or indirect in altering the pattern of development. These responses may differ
from one phenological stage to another. For example, long-term
effects of heat stress on developing seeds may include delayed
germination or loss of vigor, ultimately leading to reduced
emergence and seedling establishment. Under diurnally varying temperatures, coleoptile growth in maize reduced at 40 C
and ceased at 45 C (Weaich et al., 1996). High temperatures
caused significant declines in shoot dry mass, relative growth
rate and net assimilation rate in maize, pearl millet and sugarcane, though leaf expansion was minimally affected (Ashraf and
Hafeez, 2004; Wahid, 2007). Major impact of high temperatures
on shoot growth is a severe reduction in the first internode length
resulting in premature death of plants (Hall, 1992). For example, sugarcane plants grown under high temperatures exhibited
smaller internodes, increased tillering, early senescence, and
reduced total biomass (Ebrahim et al., 1998).
Heat stress, singly or in combination with drought, is a common constraint during anthesis and grain filling stages in many
cereal crops of temperate regions. For example, heat stress
lengthened the duration of grain filling with reduction in kernel growth leading to losses in kernel density and weight by up
to 7% in spring wheat (Guilioni et al., 2003). Similar reductions
occurred in starch, protein and oil contents of the maize kernel
(Wilhelm et al., 1999) and grain quality in other cereals under
heat stress (Maestri et al., 2002). In wheat, both grain weight
and grain number appeared to be sensitive to heat stress, as the
number of grains per ear at maturity declined with increasing
temperature (Ferris et al., 1998). In temperate and tropical lowlands, heat susceptibility is a cause of yield loss in common bean,
Phaseolus vulgaris (Rainey and Griffiths, 2005) and groundnut,
Arachis hypogea (Vara Prasad et al., 1999). In tomato, reproductive processes were adversely affected by high temperature,
which included meiosis in both male and female organs, pollen
germination and pollen tube growth, ovule viability, stigmatic
and style positions, number of pollen grains retained by the
stigma, fertilization and post-fertilization processes, growth of
the endosperm, proembryo and fertilized embryo (for a review
see (Foolad, 2005). Also, the most noticeable effect of high temperatures on reproductive processes in tomato is the production
of an exserted style (i.e., stigma is elongated beyond the anther
cone), which may prevent self-pollination. Poor fruit set at high
temperature has also been associated with low levels of carbohydrates and growth regulators released in plant sink tissues (Kinet
and Peet, 1997). Growth chamber and greenhouse studies suggest that high temperature is most deleterious when flowers are
first visible and sensitivity continues for 1015 days. Reproductive phases most sensitive to high temperature are gametogenesis
(89 days before anthesis) and fertilization (13 days after anthesis) in various plants (Foolad, 2005). Both male and female
gametophytes are sensitive to high temperature and response
varies with genotype; however, ovules are generally less heat
sensitive than pollen (Peet and Willits, 1998). Overall, based
on the available studies, it seems that plant responses to high
temperature vary with plant species and phenological stages.
Reproductive processes are markedly affected by high temper-

atures in most plants, which ultimately affect fertilization and


post-fertilization processes leading to reduced crop yield.
3.1.2. Anatomical changes
Although limited details are available, anatomical changes
under high ambient temperatures are generally similar to those
under drought stress. At the whole plant level, there is a general
tendency of reduced cell size, closure of stomata and curtailed
water loss, increased stomatal and trichomatous densities, and
greater xylem vessels of both root and shoot (Anon et al.,
2004). In grapes (Vitis vinifera), heat stress severely damaged
the mesophyll cells and increased permeability of plasma membrane (Zhang et al., 2005). With the onset of high temperature
regime, Zygophyllum qatarense produced polymorphic leaves
and tended to reduce transpirational water loss by showing
bimodal stomatal behavior (Sayed, 1996). At the sub-cellular
level, major modifications occur in chloroplasts, leading to
significant changes in photosynthesis. For example, high temperatures reduced photosynthesis by changing the structural
organization of thylakoids (Karim et al., 1997). Studies have
revealed that specific effects of high temperatures on photosynthetic membranes result in the loss of grana stacking or its
swelling. In response to heat stress, chloroplasts in the mesophyll cells of grape plants became round in shape, the stroma
lamellae became swollen, and the contents of vacuoles formed
clumps, whilst the cristae were disrupted and mitochondria
became empty (Zhang et al., 2005). Such changes result in the
formation of antenna-depleted photosystem-II (PSII) and hence
reduced photosynthetic and respiratory activities (Zhang et al.,
2005). In general, it is evident that high temperature considerably affects anatomical structures not only at the tissue and
cellular levels but also at the sub-cellular level. The cumulative
effects of all these changes under high temperature stress may
result in poor plant growth and productivity.
3.1.3. Phenological changes
Observation of changes in plant phenology in response to
heat stress can reveal a better understanding of interactions
between stress atmosphere and the plant. Different phenological stages differ in their sensitivity to high temperature; however,
this depends on species and genotype as there are great interand intra-specific variations (Wollenweber et al., 2003; Howarth,
2005). Heat stress is a major factor affecting the rate of plant
development, which may be increasing to a certain limit and
decreasing afterwards (Hall, 1992; Marcum, 1998; Howarth,
2005).
The developmental stage at which the plant is exposed to the
stress may determine the severity of possible damages experienced by the crop. It is, however, unknown whether damaging
effects of heat episodes occurring at different developmental
stages are cumulative (Wollenweber et al., 2003). Vulnerability
of species and cultivars to high temperatures may vary with the
stage of plant development, but all vegetative and reproductive
stages are affected by heat stress to some extent. During vegetative stage, for example, high day temperature can damage leaf
gas exchange properties. During reproduction, a short period
of heat stress can cause significant increases in floral buds and

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opened flowers abortion; however, there are great variations in


sensitivity within and among plant species (Guilioni et al., 1997;
Young et al., 2004). Impairment of pollen and anther development by elevated temperatures is another important factor
contributing to decreased fruit set in many crops at moderateto-high temperatures (Peet et al., 1998; Sato et al., 2006). The
staple cereal crops can tolerate only narrow temperature ranges,
which if exceeded during the flowering phase can damage fertilization and seed production, resulting in reduced yield (Porter,
2005). Under high temperature conditions, earlier heading is
advantageous in the retention of more green leaves at anthesis,
leading to a smaller reduction in yield (Tewolde et al., 2006).
Furthermore, high temperatures during grain filling can modify
flour and bread quality and other physico-chemical properties
of grain crops such as wheat (Perrotta et al., 1998), including
changes in protein content of the flour (Wardlaw et al., 2002).
Thus, for crop production under high temperatures, it is important to know the developmental stages and plant processes that
are most sensitive to heat stress, as well as whether high day
or high night temperatures are more injurious. Such insights
are important in determining heat-tolerance potential of crop
plants.
3.2. Physiological responses
3.2.1. Waters relations
Plant water status is the most important variable under changing ambient temperatures (Mazorra et al., 2002). In general,
plants tend to maintain stable tissue water status regardless of
temperature when moisture is ample; however, high temperatures severely impair this tendency when water is limiting
(Machado and Paulsen, 2001). Under field conditions, high
temperature stress is frequently associated with reduced water
availability (Simoes-Araujo et al., 2003). In Lotus creticus, for
example, elevated night temperatures caused a greater reduction in leaf water potential of water-stressed as compared to
non-stressed plants (Anon et al., 2004). In sugarcane, leaf water
potential and its components were changed upon exposure to
heat stress even though the soil water supply and relative humidity conditions were optimal, implying an effect of heat stress on
root hydraulic conductance (Wahid and Close, 2007). Similarly,
in tomato heat stress perturbed the leaf water relations and root
hydraulic conductivity (Morales et al., 2003). In general, during daytime enhanced transpiration induces water deficiency in
plants, causing a decrease in water potential and leading to perturbation of many physiological processes (Tsukaguchi et al.,
2003). High temperatures seem to cause water loss in plants
more during daytime than nighttime.
3.2.2. Accumulation of compatible osmolytes
A key adaptive mechanism in many plants grown under
abiotic stresses, including salinity, water deficit and extreme
temperatures, is accumulation of certain organic compounds
of low molecular mass, generally referred to as compatible
osmolytes (Hare et al., 1998; Sakamoto and Murata, 2002).
Under stress, different plant species may accumulate a variety of
osmolytes such as sugars and sugar alcohols (polyols), proline,

203

tertiary and quaternary ammonium compounds, and tertiary sulphonium compounds (Sairam and Tyagi, 2004). Accumulation
of such solutes may contribute to enhanced stress tolerance of
plants, as briefly described in below.
Glycinebetaine (GB), an amphoteric quaternary amine, plays
an important role as a compatible solute in plants under various
stresses, such as salinity or high temperature (Sakamoto and
Murata, 2002). Capacity to synthesize GB under stress conditions differs from species to species (Ashraf and Foolad, 2007).
For example, high level of GB accumulation was reported in
maize (Quan et al., 2004) and sugarcane (Wahid and Close,
2007) due to desiccating conditions of water deficit or high temperature. In contrast, plant species such as rice (Oryza sativa),
mustard (Brassica spp.), Arabidopsis (Arabidopsis thaliana) and
tobacco (Nicotiana tabacum) naturally do not produce GB under
stress conditions. However, genetic engineering has allowed
the introduction of GB-biosynthetic pathways into GB-deficit
species (Sakamoto and Murata, 2002; Quan et al., 2004).
Like GB, proline is also known to occur widely in higher
plants and normally accumulates in large quantities in response
to environmental stresses (Kavi Kishore et al., 2005). In assessing the functional significance of accumulation of compatible
solutes, it is suggested that proline or GB synthesis may buffer
cellular redox potential under heat and other environmental
stresses (Wahid and Close, 2007). Similarly, accumulation of
soluble sugars under heat stress has been reported in sugarcane,
which entails great implications for heat tolerance (Wahid and
Close, 2007). Under high temperatures, fruit set in tomato plants
failed due to the disruption of sugar metabolism and proline
transport during the narrow window of male reproductive development (Sato et al., 2006). Hexose sensing in transgenic plants
engineered to produce trehalose, fructans or mannitol may be an
important contributory factor to the stress-tolerant phenotypes
(Hare et al., 1998).
Among other osmolytes, -4-aminobutyric acid (GABA), a
non-protein amino acid, is widely distributed throughout the biological world to act as a compatible solute. GABA is synthesized
from the glutamic acid by a single step reaction catalyzed by glutamate decarboxylase (GAD). An acidic pH activates GAD, a
key enzyme in the biosynthesis of GABA. Episodes of high
tempeartures increase the cytosolic level of Ca, which leads
to calmodulin-mediated activation of GAD (Taiz and Zeiger,
2006). Several other studies show that various environmental stresses increase GABA accumulation through metabolic or
mechanical disruptions, thus leading to cytosolic acidification.
Kinetics of GABA in plants show a stress-specific pattern of
accumulation, which is consistent with its physiological role in
the mitigation of stress effects. Rapid accumulation of GABA in
stressed tissues may provide a critical link in the chain of events
stemming from perception of environmental stresses to timely
physiological responses (Kinnersley and Turano, 2000).
In summary, because of significant roles of osmolytes in
response to environmenal stresses in plants, crop stress (e.g.,
heat) tolerance might be enhanced by increased accumulation of compatible solutes through traditional plant breeding,
marker-assisted selection (MAS) or genetic engineering (GE)
approaches (for a review see (Ashraf and Foolad, 2007).

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3.2.3. Photosynthesis
Alterations in various photosynthetic attributes under heat
stress are good indicators of thermotolerance of the plant as they
show correlations with growth. Any constraint in photosynthesis can limit plant growth at high temperatures. Photochemical
reactions in thylakoid lamellae and carbon metabolism in the
stroma of chloroplast have been suggested as the primary sites
of injury at high temperatures (Wise et al., 2004). Chlorophyll
fluorescence, the ratio of variable fluorescence to maximum
fluorescence (Fv /Fm ), and the base fluorescence (F0 ) are physiological parameters that have been shown to correlate with heat
tolerance (Yamada et al., 1996). Increasing leaf temperatures
and photosynthetic photon flux density influence thermotolerance adjustments of PSII, indicating their potential to optimise
photosynthesis under varying environmental conditions as long
as the upper thermal limits do not exceed (Salvucci and CraftsBrandner, 2004b; Marchand et al., 2005). In tomato genotypes
differing in their capacity for thermotolerance as well as in
sugarcane, an increased chlorophyll a:b ratio and a decreased
chlorophyll:carotenoids ratio were observed in the tolerant genotypes under high temperatures, indicating that these changes
were related to thermotolerance of tomato (Camejo et al., 2005;
Wahid and Ghazanfar, 2006). Furthermore, under high temperatures, degradation of chlorophyll a and b was more pronounced
in developed compared to developing leaves (Karim et al., 1997,
1999). Such effects on chlorophyll or photosynthetic apparatus
were suggested to be associated with the production of active
oxygen species (Camejo et al., 2006; Guo et al., 2006).
PSII is highly thermolabile, and its activity is greatly reduced
or even partially stopped under high temperatures (Bukhov et
al., 1999; Camejo et al., 2005), which may be due to the properties of thylakoid membranes where PSII is located (Mcdonald
and Paulsen, 1997). Heat stress may lead to the dissociation
of oxygen evolving complex (OEC), resulting in an imbalance
between the electron flow from OEC toward the acceptor side of
PSII in the direction of PSI reaction center (Fig. 1) (De Ronde
et al., 2004). Heat stress causes dissociation of a manganese
(Mn)-stabilizing 33-kDa protein at PSII reaction center complex followed by the release of Mn atoms (Yamane et al., 1998).
Heat stress may also impair other parts of the reaction center,
e.g., the D1 and/or the D2 proteins (De Las Rivas and Barber,
1997). In wheat, high temperatures and excessive light dam-

Fig. 1. Heat induced inhibition of oxygen evolution and PSII activity. Heat stress
leads to either (1) dissociation or (2) inhibition of the oxygen evolving complexes
(OEC). This enables an alternative internal e -donor such as proline instead of
H2 O to donate electrons to PSII. Reproduced with permission from De Ronde
et al. (2004).

aged different sites of PSII, which implied different pathways


for the recovery of its functional activity (Sharkova, 2001). In
barley, heat pulses abruptly damaged the PSII units and caused
loss of their capacity of oxygen evolution leading to a restricted
electron transport, which was totally abolished after four hours
(Toth et al., 2005). This implied that the degradation of the
impaired PSII units occurred in the light during this period of
time. Following this, de novo synthesis of PSII units in the light
gave a gradual rise to the observed PSII activities. These effects
can result from different events, including inhibition of electron
transport activity and limited generation of reducing powers for
metabolic functions (Allakhverdieva et al., 2001). In field-grown
Pima cotton under high temperatures, leaf photosynthesis was
functionally limited by photosynthetic electron transport and
ribulose-1,5-bisphosphate (RuBP) regeneration capacity, but not
rubisco activity (Wise et al., 2004). On the other hand, under high
temperatures, PSI stromal enzymes and chloroplast envelops
are thermostable and in fact PSI driven cyclic electron pathway,
capable of contributing to thylakoid proton gradient, is activated
(Bukhov et al., 1999).
High temperature influences the photosynthetic capacity of
C3 plants more strongly than in C4 plants. It alters the energy
distribution and changes the activities of carbon metabolism
enzymes, particularly the rubisco, thereby altering the rate of
RuBP regeneration by the disruption of electron transport and
inactivation of the oxygen evolving enzymes of PSII (Salvucci
and Crafts-Brandner, 2004b). Heat shock reduces the amount of
photosynthetic pigments (Todorov et al., 2003), soluble proteins,
rubisco binding proteins (RBP) and large- (LS) and smallsubunits (SS) of rubisco in darkness but increases them in light,
indicating their roles as chaperones and HSPs (Kepova et al.,
2005). Moreover, under heat stress, starch or sucrose synthesis is greatly influenced as observed from reduced activities
of sucrose phosphate synthase (Chaitanya et al., 2001), ADPglucose pyrophosphorylase and invertase (Vu et al., 2001).
In any plant species, the ability to sustain leaf gas exchange
under heat stress has a direct relationship with heat tolerance.
During the vegetative stage, high day temperature can cause
damage to compensated leaf photosynthesis, reducing CO2
assimilation rates (Hall, 1992). Increased temperatures curtail
photosynthesis and increase CO2 transfer conductance between
intercellular spaces and carboxylation sites. Stomatal conductance (gs) and net photosynthesis (Pn) are inhibited by moderate
heat stress in many plant species due to decreases in the activation state of rubisco (Crafts-Brander and Salvucci, 2002;
Morales et al., 2003). Although with an increase in temperature
rubisco catalytic activity increases, a low affinity of the enzyme
for CO2 and its dual nature as an oxygenase limits the possible
increases in Pn. For example, in maize the Pn was inhibited at leaf
temperatures above 38 C and inhibition was much more severe
when temperature was increased abruptly rather than gradually.
However, this inhibition was independent of stomatal response to
high temperature (Crafts-Brander and Salvucci, 2002). Despite
observed negative effects of high temperature, the optimum temperature for leaf photosynthesis is likely to increase with elevated
levels of atmospheric CO2 . Several studies have concluded that
CO2 -induced increases in crop yields are much more plausi-

A. Wahid et al. / Environmental and Experimental Botany 61 (2007) 199223

ble in warm- than in cool-season crops. Thus, despite its other


potential negative implications, global warming may not greatly
affect the overall Pn.
A well-known consequence of elevated temperature in plants
is the damage caused by heat-induced imbalance in photosynthesis and respiration; in general the rate of photosynthesis
decreases while dark- and photo-respiration rates increase considerably under high temperatures. Also, rate of biochemical
reactions decreases and enzyme inactivation and denaturation
take place as the temperature increases leading to severely
reduced photosynthesis (Nakamoto and Hiyama, 1999). However, the magnitude of such alterations in response to heat stress
differs with species and genotypes (Mcdonald and Paulsen,
1997). Furthermore, it has been determined that the photosynthetic CO2 assimilation rate is less affected by heat stress
in developing leaves than in completely developed leaves.
Heat stress normally decreases the duration of developmental phases leading to smaller organs, reduced light perception
and carbon assimilation processes including transpiration,
photosynthesis and respiration (Stone, 2001). Nonetheless, photosynthesis is considered as the physiological process most
sensitive to high temperatures, and that rising atmospheric
CO2 content will drive temperature increases in many already
stressful environments. This CO2 -induced increase in plant
high-temperature tolerance may have a substantial impact on
both the productivity and distribution of many crop species in
future.
3.2.4. Assimilate partitioning
Under low to moderate heat stress, a reduction in source and
sink activities may occur leading to severe reductions in growth,
economic yield and harvest index. Assimilate partitioning, taking place via apoplastic and symplastic pathways under high
temperatures, has significant effects on transport and transfer
processes in plants (Taiz and Zeiger, 2006). However, considerable genotypic variation exists in crop plants for assimilate
partitioning, as for example among wheat genotypes (Yang et
al., 2002). To elucidate causal agents of reduced grain filling
in wheat under high temperatures, Wardlaw (1974) examined
three main components of the plant system including source
(flag leaf blade), sink (ear), and transport pathway (peduncle).
It was determined that photosynthesis had a broad temperature
optimum from 20 to 30 C, however it declined rapidly at temperatures above 30 C. The rate of 14 C assimilate movement out
of the flag leaf (phloem loading), was optimum around 30 C,
however, the rate of movement through the stem was independent of temperature from 1 to 50 C. It was concluded that, at
least in wheat, temperature effects on translocation result indirectly from temperature effects on source and sink activities.
From such results, increased mobilization efficiency of reserves
from leaves, stem or other plant parts has been suggested as a
potential strategy to improve grain filling and yield in wheat
under heat stress. This suggestion, however, is based on present
limited knowledge of physiological basis of assimilate partitioning under high temperature stress. Further investigation in
this area may lead to improved crop production efficiency under
high-temperature stress.

205

3.2.5. Cell membrane thermostability


Sustained function of cellular membranes under stress is pivotal for processes such as photosynthesis and respiration (Blum,
1988). Heat stress accelerates the kinetic energy and movement
of molecules across membranes thereby loosening chemical
bonds within molecules of biological membranes. This makes
the lipid bilayer of biological membranes more fluid by either
denaturation of proteins or an increase in unsaturated fatty acids
(Savchenko et al., 2002). The integrity and functions of biological membranes are sensitive to high temperature, as heat stress
alters the tertiary and quaternary structures of membrane proteins. Such alterations enhance the permeability of membranes,
as evident from increased loss of electrolytes. The increased
solute leakage, as an indication of decreased cell membrane thermostability (CMT), has long been used as an indirect measure
of heat-stress tolerance in diverse plant species, including soybean (Martineau et al., 1979), potato and tomato (Chen et al.,
1982), wheat (Blum et al., 2001), cotton (Ashraf et al., 1994),
sorghum (Marcum, 1998), cowpea (Ismail and Hall, 1999) and
barley (Wahid and Shabbir, 2005). Electrolyte leakage is influenced by plant/tissue age, sampling organ, developmental stage,
growing season, degree of hardening and plant species. In maize,
injuries to plasmalemma due to heat stress were much less severe
in developing than in mature leaves (Karim et al., 1997, 1999). It
was determined that an increase in saturated fatty acids in mature
leaves elevated melting temperature of plasma membranes and
thus reducing heat tolerance of the plant. In Arabidopsis plants
grown under high temperature, total lipid content in membranes
decreased to about one-half and the ratio of unsaturated to saturated fatty acids decreased to one-third of the levels at normal
temperatures (Somerville and Browse, 1991). It should be noted,
however, that in some species heat tolerance does not correlate
with the degree of lipid saturation, suggesting that factors other
than membrane stability might be limiting the growth at high
temperatures.
The relationship between CMT and crop yield under high
temperatures may vary from plant to plant and invokes for study
of individual crops before using it as an important physiological
selection criterion. For example, whereas a significant relationship between CMT and yield was observed in a few plant species
such as sorghum (Sullivan and Ross, 1979), no such relationship was observed in soybean (Martineau et al., 1979) or wheat
(Shanahan et al., 1990). Thus, the major cause(s) of yield suppression under heat stress remain largely elusive and deserve
further experimentation.
3.2.6. Hormonal changes
Plants have the ability to monitor and adapt to adverse
environmental conditions, though the degree of adaptability or
tolerance to specific stresses varies among species and genotypes. Hormones play an important role in this regard. Cross-talk
in hormone signaling reflects an organisms ability to integrate
different inputs and respond appropriately. Hormonal homeostasis, stability, content, biosynthesis and compartmentalization are
altered under heat stress (Maestri et al., 2002).
Abscisic acid (ABA) and ethylene (C2 H4 ), as stress hormones, are involved in the regulation of many physiological

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properties by acting as signal molecules. Different environmental stresses, including high temperature, result in increased levels
of ABA. For example, recently it was determined that in creeping bentgrass (Agrostis palustris), ABA level did not rise during
heat stress, but it accumulated upon recovery from stress suggesting a role during the latter period (Larkindale and Huang,
2005). However, the action of ABA in response to stress involves
modification of gene expression. Analysis of ABA-responsive
promoters revealed several potential cis- and trans-acting regulatory elements (Swamy and Smith, 1999). ABA mediates
acclimation/adaptation of plants to desiccation by modulating
the up- or down-regulation of numerous genes (Xiong et al.,
2002). Under field conditions, where heat and drought stresses
usually coincide, ABA induction is an important component
of thermotolerance, suggesting its involvement in biochemical
pathways essential for survival under heat-induced desiccation
stress (Maestri et al., 2002). Other studies also suggest that
induction of several HSPs (e.g., HSP70) by ABA may be one
mechanism whereby it confers thermotolerance (Pareek et al.,
1998). More so, heat shock transcription factor 3 acts synergistically with chimeric genes with a small HSP promoter, which
is ABA inducible (Rojas et al., 1999).
A gaseous hormone, ethylene regulates almost all growth
and developmental processes in plants, ranging from seed germination to flowering and fruiting as well as tolerance to
environmental stresses. Measurement of the rate of ethylene
released per unit amount of tissue provides information on the
relative changes in cellular concentration of C2 H4 . Ethylene
has nearly full biological activity at 1 L L1 , corresponding
to 6.5 109 M at 25 C. However, the levels of ethylene or
the enzymes involved in ethylene biosynthesis vary at different
time intervals during the day. For instance, the endogenous concentration of 1-amino-cyclopropane-1-carboxylic acid (ACC), a
precursor of ethylene biosynthesis, measured at predawn and at
maximum solar radiation during a summer drought in rosemary
(Rosmarinus ofcinalis) showed a sharp distinction between the
two times, which was positively correlated with the intensity of
incident solar radiations (Munne-Bosch et al., 2002).
Heat stress changes ethylene production differently in different plant species (Arshad and Frankenberger, 2002). For
example, while ethylene production in wheat leaves was inhibited slightly at 35 C and severely at 40 C, in soybean ethylene
production in hypocotyls increased by increasing temperature
up to 40 C and it showed inhibition at 45 C. Despite the fact
that ACC accumulated in both species at 40 C, its conversion
into ethylene occurred only in soybean hypocotyls but not in
wheat. Wheat leaves transferred to 18 C followed by a short
exposure to 40 C showed an increase in ethylene production
after 1 h lag period, possibly due to conversion of accumulated
ACC to ethylene during that period (Tan et al., 1988). Similarly,
creeping bentgrass showed ethylene production upon recovery,
but not when under heat stress (Larkindale and Huang, 2005).
Temperatures up to 35 C have been shown to increase ethylene production and ripening of propylene-treated kiwifruit, but
temperature above 35 C inhibits ripening by inhibiting ethylene
production, although respiration continues until the tissue disintegration (Antunes and Sfakiotakis, 2000). In pepper (Piper

nigrum), increase in the level of ACC was positively correlated with high temperatures (Huberman et al., 1997). Exposure
of imbibed sunflower seed to 45 C for 48 h induced a state
of thermodormancy, which appeared to associate with the loss
of seeds ability to convert ACC to ethylene (Corbineau et al.,
1989). However, treatment with 2.5 mM ethephon or 55 L L1
ethylene improved germination of the seed at 25 C. Ethylene
may overcome the inhibitory effect of high temperature on thermosensitive lettuce seed due to increased -mannanase activity,
which helps weakening of the endosperm and facilitates germination (Nascimento et al., 2004). High temperature-induced
abscission of reproductive organs relates to an increased ACC
level; this is accompanied with both reduced levels and transport capacity of auxins to reproductive organs (Huberman et al.,
1997). The effect of pre-harvest temperature on ripening characteristics of shaded and sun-exposed apple fruits indicated that
the former treatment produced up to 90% less ethylene than the
latter (Klein et al., 2001). In maize, ethylene production was
highest at the top ear and lowest at the middle ear, suggesting
that ethylene plays an important role in assimilate partitioning
to grain filling (Fenglu et al., 1997).
Among other hormones, salicylic acid (SA) has been suggested to be involved in heat-stress responses elicited by plants.
SA is an important component of signaling pathways in response
to systemic acquired resistance (SAR) and the hypersensitive
response (HR) (Kawano et al., 1998). SA stabilizes the trimers
of heat shock transcription factors and aids them bind heat shock
elements to the promoter of heat shock related genes. Longterm thermotolerance can be induced by SA, in which both
Ca2+ homeostasis and antioxidant systems are thought to be
involved (Wang and Li, 2006b). Sulphosalicylic acid (SSA), a
derivative of SA, treatment can effectively remove H2 O2 and
increase heat tolerance. In this regard, catalase (CAT) plays
a key role in removing H2 O2 in cucumber (Cucumus sativus)
seedlings treated with SSA under heat stress. In contrast, while
glutathione peroxidase (GPX), ascorbate peroxidase (APX) and
glutathione reductase (GR) showed higher activities in all SSA
treatments under heat stress, they were not key enzymes in
removing H2 O2 (Shi et al., 2006). Thermotolerance of plants
also can be enhanced by spraying leaves with acetyl-SA (Dat
et al., 1998). Likewise, methyl salicylate (MeSA), a derivative
of SA, has multiple functions. In addition to acting as signal
molecule, it gives thermotolerance to holm oak (Quercus ilex)
by enhanced xanthophylls de-epoxidation and content of ascorbate, antioxidants and -tocopherol in leaves (Llusia et al., 2005;
Wang and Li, 2006a).
The effects of gibberellins and cytokinins on high temperature tolerance are opposite to that of ABA. An inherently
heat-tolerant dwarf mutant of barley impaired in the synthesis of gibberellins was repaired by application of gibberellic
acid, whereas application of triazole paclobutrazol, a gibberellin
antagonist, conferred heat tolerance (Vettakkorumakankav et al.,
1999). In creeping bentgrass, the levels of various cytokinins,
zeatin (Z), zeatin riboside (ZR), dihydrogen zeatin riboside
(DHZR) and isopentinyl adenosine (iPA), showed dramatic
decreases by the 5th day in root and 10th day in shoot,
which were correlated with decreased dry matter production

A. Wahid et al. / Environmental and Experimental Botany 61 (2007) 199223

(Liu and Huang, 2005). In a dwarf wheat variety, hightemperature-induced decrease in cytokinin content was found
to be responsible for reduced kernel filling and its dry weight
(Banowetz et al., 1999). Another class of hormones, brassinosteroids have recently been shown to confer thermotolerance to
tomato and oilseed rape (Brassica napus), but not to cereals
(Dhaubhadel et al., 1999). The potential roles of other phytohormones in plant thermotolerance are yet unknown.
3.2.7. Secondary metabolites
Most of the secondary metabolites are synthesized
from the intermediates of primary carbon metabolism via
phenylpropanoid, shikimate, mevalonate or methyl erythritol
phosphate (MEP) pathways (Wahid and Ghazanfar, 2006).
High-temperature stress induces production of phenolic compounds such as flavonoids and phenylpropanoids. Phenylalanine
ammonia-lyase (PAL) is considered to be the principal enzyme
of the phenylpropanoid pathway. Increased activity of PAL in
response to thermal stress is considered as the main acclimatory response of cells to heat stress. Thermal stress induces the
biosynthesis of phenolics and suppresses their oxidation, which
is considered to trigger the acclimation to heat stress for example
as in watermelon, Citrulus vulgaris (Rivero et al., 2001).
Carotenoids are widely known to protect cellular structures
in various plant species irrespective of the stress type (Havaux,
1998; Wahid and Ghazanfar, 2006; Wahid, 2007). For example,
the xanthophyll cycle (the reversible interconversion of two particular carotenoids, violaxanthin and zeaxanthin) has evolved
to play this essential role in photoprotection. Since zeaxanthin
is hydrophobic, it is found mostly at the periphery of the lightharvesting complexes, where it functions to prevent peroxidative
damage to the membrane lipids triggered by ROS (Horton,
2002). Recent studies have revealed that carotenoids of the xanthophyll family and some other terpenoids, such as isoprene or
-tocopherol, stabilize and photoprotect the lipid phase of the
thylakoid membranes (Havaux, 1998; Sharkey, 2005; Velikova
et al., 2005). When plants are exposed to potentially harmful
environmental conditions, such as strong light and/or elevated
temperatures, the xanthophylls including violaxanthin, antheraxanthin and zeaxanthin partition between the light-harvesting
complexes and the lipid phase of the thylakoid membranes. The
resulting interaction of the xanthophyll molecules and the membrane lipids brings about a decreased fluidity (thermostability)
of membrane and a lowered susceptibility to lipid peroxidation
under high temperatures (Havaux, 1998).
Phenolics, including flavonoids, anthocyanins, lignins, etc.,
are the most important class of secondary metabolites in plants
and play a variety of roles including tolerance to abiotic stresses
(Chalker-Scott, 2002; Wahid and Ghazanfar, 2006; Wahid,
2007). Studies suggest that accumulation of soluble phenolics under heat stress was accompanied with increased phenyl
ammonia lyase (PAL) and decreased peroxidase and polyphenol
lyase activities (Rivero et al., 2001). Anthocyanins, a subclass
of flavonoid compounds, are greatly modulated in plant tissues
by prevailing high temperature; low temperature increases and
elevated temperature decreases their concentration in buds and
fruits (Sachray et al., 2002). For example, high temperature

207

decreases synthesis of anthocyanins in reproductive parts of red


apples (Tomana and Yamada, 1988), chrysanthemums (Shibata
et al., 1988) and asters (Sachray et al., 2002). One of the causes
of low anthocyanin concentration in plants at high temperatures
is a decreased rate of its synthesis and stability (Sachray et al.,
2002). On the other hand, vegetative tissues under high temperature stress show an accumulation of anthocyanins including
rose and sugarcane leaves (Wahid and Ghazanfar, 2006). It has
been suggested that in addition to their role as UV screen,
anthocyanins serve to decrease leaf osmotic potential, which
is linked to increased uptake and reduced transpirational loss of
water under environmental stresses including high temperature
(Chalker-Scott, 2002). These properties may enable the leaves
to respond quickly to changing environmental conditions.
Isoprenoids, another class of plant secondary products, are
synthesized via mevalonate pathway (Taiz and Zeiger, 2006).
Being of low molecular weight and volatile in nature, their
emission from leaves has been reported to confer heat-stress
tolerance to photosynthesis apparati in different plants (Loreto
et al., 1998). Studies have revealed that their biosynthesis is
cost effective. While deriving considerable amount of photosynthates, they show compensatory benefits as to heat tolerance
(Funk et al., 2004). Plants capable of emitting greater amounts
of isoprene generally display better photosynthesis under heat
stress, thus there is a relationship between isoprene emission
and heat-stress tolerance (Velikova and Loreto, 2005). Sharkey
(2005) opined that isoprene production protects the PSII from
the damage caused by ROS, including H2 O2 , produced during
heat-induced oxygenase action of rubisco, even though the photosynthetic rate approaches zero. It is proposed that endogenous
production of isoprene protects the biological membranes from
damaging effects by directly reacting with oxygen singlets (1 O2 )
by means of isoprene-conjugate double bond (Velikova et al.,
2005).
In summary, like other stresses, heat stress causes accumulation of secondary metabolites of multifarious nature in plants.
However, the specific roles they play in enhancing heat-stress
tolerance seem to be different and warrant further elucidation.
3.3. Molecular responses
3.3.1. Oxidative stress and antioxidants
In addition to tissue dehydration, heat stress may induce
oxidative stress. For example, generation and reactions of activated oxygen species (AOS) including singlet oxygen (1 O2 ),
superoxide radical (O2 ), hydrogen peroxide (H2 O2 ) and
hydroxyl radical (OH ) are symptoms of cellular injury due
to high temperature (Liu and Huang, 2000). AOS cause the
autocatalytic peroxidation of membrane lipids and pigments
thus leading to the loss of membrane semi-permeability and
modifying its functions (Xu et al., 2006).
Superoxide radical is regularly synthesized in the chloroplast
and mitochondrion and some quantities are also produced in
microbodies (Fig. 2). The scavenging of O2 by superoxide
dismutase (SOD) results in the production of H2 O2 , which is
removed by APX or CAT. However, both O2 and H2 O2 are not
as toxic as the (OH ), which is formed by the combination of

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A. Wahid et al. / Environmental and Experimental Botany 61 (2007) 199223

Fig. 2. Schematic presentation for generation and scavenging of superoxide radical, hydrogen peroxide, hydroxyl radical-induced lipid peroxidation and glutathione
peroxidase-mediated fatty acid stabilization under environmental stresses. APX, ascorbate peroxidase; ASC, ascorbate; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; Fd, ferredoxin; GR, glutathione reductase; GSH, red glutathione; GSSG, oxi-glutathione; HO, hydroxyl radical; LH, lipid; L, LOO; LOOH,
unstable lipid radicals and hydroperoxides; LOH, stable lipid (fatty acid); MDHA, monodehydro-ascorbate; MDHAR, mono dehydro-ascorbate reductase; NE,
non-enzymatic reaction; PHGPX, phospholipid-hydroperoxide glutathione peroxidase; SOD, superoxide dismutase. Reproduced with permission from Sairam and
Tyagi (2004).

O2 and H2 O2 in the presence of trace amounts of Fe2+ and Fe3+


by the HaberWeiss reaction. The OH can damage chlorophyll,
protein, DNA, lipids and other important macromolecules, thus
fatally affecting plant metabolism and limiting growth and yield
(Sairam and Tyagi, 2004).
As depicted in Fig. 2, plants have developed a series of
both enzymatic and non-enzymatic detoxification systems to
counteract AOS, thereby protecting cells from oxidative damage (Sairam and Tyagi, 2004). For example, overexpression
of SOD in plants affect a number of physiological phenomena, which include the removal of H2 O2 , oxidation of toxic
reductants, biosynthesis and degradation of lignin in cell walls,
auxin catabolism, defensive responses to wounding, defense
against pathogen or insect attack, and some respiratory processes
(Scandalios, 1993). More specifically, expression and activation
of APX is related to the appearance of physiological injuries
caused in plants by thermal stress (Mazorra et al., 2002).
Decrease in antioxidant activity in stressed tissues results in
higher levels of AOS that may contribute to injury (Fadzillah
et al., 1996). Protection against oxidative stress is an important
component in determining the survival of a plant under heat
stress. Studies on heat-acclimated versus non-acclimated cool
season turfgrass species suggested that the former had lower
production of ROS as a result of enhanced synthesis of ascorbate and glutathione (Xu et al., 2006). Available data suggest that
some signaling molecules may cause an increase in the antioxidant capacity of cells (Gong et al., 1997; Dat et al., 1998).
Certainly further research is necessary to identify the signaling
molecules, which enhanced production of antioxidants in cells
exposed to heat stress.

3.3.2. Stress proteins


Expression of stress proteins is an important adaptation to
cope with environmental stresses. Most of the stress proteins
are soluble in water and therefore contribute to stress tolerance presumably via hydration of cellular structures (Wahid and
Close, 2007). Although heat shock proteins (HSPs) are exclusively implicated in heat-stress response, certain other proteins
are also involved.
3.3.2.1. Heat shock proteins. Synthesis and accumulation of
specific proteins are ascertained during a rapid heat stress, and
these proteins are designated as HSPs. Increased production of
HSPs occurs when plants experience either abrupt or gradual
increase in temperature (Nakamoto and Hiyama, 1999; Schoffl
et al., 1999). Induction of HSPs seems to be a universal response
to temperature stress, being observed in all organisms ranging
from bacteria to human (Vierling, 1991). Plants of arid and semiarid regions may synthesize and accumulate substantial amounts
of HSPs. Certain HSPs are also expressed in some cells under
cyclic or developmental control (Hopf et al., 1992). In this case,
the expression of HSPs is restricted to certain stages of development, such as embryogenesis, germination, pollen development
and fruit maturation (Prasinos et al., 2005). In higher plants,
HSPs are usually induced under heat shock at any stage of
development and major HSPs are highly homologous among
distinct organisms (Vierling, 1991). HSP-triggered thermotolerance is attributed to the observations that (a) their induction
coincides with the organism under stress, (b) their biosynthesis
is extremely fast and intensive, and (c) they are induced in a
wide variety of cells and organisms.

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Three classes of proteins, as distinguished by molecular


weight, account for most HSPs, viz., HSP90, HSP70 and low
molecular weight proteins of 1530 kDa. The special importance
of small HSPs in plants is suggested by their unusual abundance and diversity. The proportions of these three classes differ
among plant species. HSP70 and HSP90 mRNAs can increase
ten-fold, while low molecular weight (LMW) HSPs can increase
as much as 200-fold. Other proteins, such as 110 kDa polypeptides and ubiquitin, though less important, are also considered
to be HSPs (Feussner et al., 1997). All small-HSPs in plants are
encoded by six nuclear gene families, each gene family corresponding to proteins found in distinct cellular compartments like
cytosol, chloroplast, endoplasmic reticulum (ER), mitochondria
and membranes. Some nuclear-encoded HSPs accumulate in
the cytosol at low (27 C) and high (43 C) temperatures, but
they accumulate in chloroplast at moderate (37 C) temperatures (Waters et al., 1996). The gene for a nuclear-encoded HSP,
Hsa32, encoding a 32 kDa protein, has been cloned in tomato
(Liu et al., 2006).
Immuno-localization studies have determined that HSPs normally associate with particular cellular structures, such as cell
wall, chloroplasts, ribosomes and mitochondria (Nieto-Sotelo
et al., 2002; Yang et al., 2006). When maize, wheat and rye
seedlings were subjected to heat shocks (42 C), whereas five
mitochondrial LMW-HSPs (28, 23, 22, 20 and 19 kDa) were
expressed in maize, only one (20 kDa) was expressed in wheat
and rye, suggesting the reason for higher heat tolerance in
maize than in wheat and rye (Korotaeva et al., 2001). In another
study, a heat-tolerant maize line (ZPBL-304) exhibited increased
amounts of chloroplast protein synthesis elongation factor under
heat stress, which was related to the development of heat tolerance (Moriarty et al., 2002). In tomato plants under heat
stress, HSPs aggregate into a granular structure in the cytoplasm, possibly protecting the protein biosynthesis machinery
(Miroshnichenko et al., 2005). Presence of HSPs can prevent
denaturation of other proteins caused by high temperature. The
conformational dynamism and aggregate state of small HSPs
may be crucial for their functions in thermoprotection of plant
cells from detrimental effects of heat stress (Schoffl et al., 1999;
Iba, 2002). The ability of small HSPs to assemble into heat
shock granules (HSGs) and their disintegration is a prerequisite
for survival of plant cells under continuous stress conditions at
sublethal temperatures (Miroshnichenko et al., 2005).
In response to high temperatures, specific HSPs have been
identified in different plant species. For example, HSP68,
which is localized in mitochondria and normally constitutively
expressed, was determined to have increased expression under
heat stress in cells of potato, maize, tomato, soybean and barley (Neumann et al., 1993). Another HSP identified in maize
is a nucleus-localized protein, HSP101, which belongs to the
campylobacter invasion antigen B (CiaB) protein sub-family,
whose members promote the renaturation of protein aggregates,
and are essential for the induction of thermotolerance. Levels of
HSP101 increased in response to heat shock, more abundantly
in developing tassel, ear, silks, endosperm and embryo and
less abundantly in vegetative and floral meristematic regions,
mature pollen, roots and leaves (Young et al., 2001). In addi-

209

tion, heat treatment increases the level of other maize HSPs,


which are associated with plant ability to withstand heat stress.
For example, A 45-kDa HSP was found to play a major role
in recovery from heat stress (Ristic and Cass, 1992). Different
studies have determined that cytosolic accumulations of nuclear
encoded chloroplast proteins were reversible (within 3 h) following return to normal growth temperature in many seed bearing
species (Heckathorn et al., 1998).
There are considerable variations in patterns of HSP production in different species and even among genotypes within
species (Wood et al., 1998). Furthermore, the ability to synthesize characteristic proteins at 40 C and the intensity and
duration of synthesis differ among various tissues examined
within the same plant. Fast accumulation of HSPs in sensitive organs/tissues can play an important role in protection of
metabolic apparati of the cell, thereby acting as a key factor for
plants adaptation to, and survival under, stress. In different plant
species, elongating segments of primary roots exhibited a strong
ability to synthesize nucleus-localized HSPs, which had roles in
thermotolerance (Nieto-Sotelo et al., 2002). Under heat-stress
conditions, while synthesis of a typical set of HSPs was induced
in male tissues of maize flowers undergoing pollen formation,
the mature pollen showed no synthesis of HSPs and thus were
sensitive to heat stress and responsible for the failure of fertilization at high temperatures (Dupuis and Dumas, 1990). In
another study, germinating maize pollen showed induction of
64 and 72 kDa peptides of HSPs under heat stress (Frova et al.,
1989) whilst in the whole plant expression of a 45 kDa HSP was
responsible for the heat tolerance (Ristic et al., 1996). Similar
variation in the expression of HSPs can be found in other plant
species.
The mechanism by which HSPs contribute to heat tolerance is
still enigmatic though several roles have been ascribed to them.
Many studies assert that HSPs are molecular chaperones insuring the native configuration and functionality of cell proteins
under heat stress. There is considerable evidence that acquisition of thermotolerance is directly related to the synthesis and
accumulation of HSPs (Bowen et al., 2002). For instance, HSPs
provide for new or distorted proteins to fold into shapes essential for their normal functions. They also help shuttling proteins
from one compartment to another and transporting old proteins
to garbage disposals inside the cell. Among others, HSP70
has been extensively studied and is proposed to have a variety of
functions such as protein translation and translocation, proteolysis, protein folding or chaperoning, suppressing aggregation, and
reactivating denatured proteins (Zhang et al., 2005). Recently,
dual role of LMW HSP21 in tomato has been described as protecting PSII from oxidative damage and involvement in fruit
color change during storage at low temperatures (Neta-Sharir et
al., 2005).
In many plant species, thermotolerance of cells and tissues
after a heat stress is pretty much dependent upon induction of
HSP70, though HSP101 has also been shown to be essential
(Schoffl et al., 1999). One hypothesis is that HSP70 participates
in ATP-dependent protein unfolding or assembly/disassembly
reactions and it prevents protein denaturation during heat stress
(Iba, 2002). Evidence for the general protective roles of HSPs

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comes from the fact that mutants unable to synthesize them


or the cells in which HSP70 synthesis is blocked or inactivated are more susceptible to heat injury (Burke, 2001). Heat
sensitivity was associated with reduced capacity of bentgrass
variants to accumulate chloroplastic HSPs (Wang and Luthe,
2003). The level of HSP22, a member of the plant small HSP
super-family, remained high under continuous heat stress (Lund
et al., 1998). LMW-HSPs may play structural roles in maintaining cell membrane integrity. Localization of LMW-HSPs in
chloroplast membranes further suggested that these proteins protect the PSII from adverse effects of heat stress and play a role
in photosynthetic electron transport (Barua et al., 2003).
3.3.2.2. Other heat induced proteins. Besides HSPs, there are
a number of other plant proteins, including ubiquitin (Sun and
Callis, 1997), cytosolic Cu/Zn-SOD (Herouart and Inze, 1994)
and Mn-POD (Brown et al., 1993), whose expressions are stimulated upon heat stress. For example, in Prosopis chilensis and
soybean under heat stress, ubiquitin and conjugated-ubiquitin
synthesis during the first 30 min of exposure emerged as an
important mechanism of heat tolerance (Ortiz and Cardemil,
2001). Some studies have shown that heat shock induces Mnperoxidase, which plays a vital role in minimizing oxidative
damages (Iba, 2002). In a study on Chenopodium murale, when
leaf proteins extracts from thylakoid and stromal fractions were
subjected to heat stress it was determined that Cu/Zn-SOD
from stromal fraction was more heat tolerant than Cu/Zn-SOD
from thylakoid, and this was responsible for chloroplastic stability under heat stress (Khanna-Chopra and Sabarinath, 2004).
In another study, a number of osmotin like proteins induced
by heat and nitrogen stresses, collectively called Pir proteins,
were found to be overexpressed in the yeast cells under heat
stress conferring them resistance to tobacco osmotin (an antifungal) (Yun et al., 1997). Late embryogenesis abundant (LEA)
proteins can prevent aggregation and protect the citrate synthase from desiccating conditions like heat- and drought-stress
(Goyal et al., 2005). Using proteomics tool, Majoul et al.
(2003) determined enhanced expressions of 25 LEA proteins in
hexaploid wheat during grain filling. Geranium leaves exposed
to drought and heat stress revealed expression of dehydrin proteins (2560 kDa), which indicated a possible linkage between
drought and heat-stress tolerance (Arora et al., 1998). Recently,
three low-molecular-weight dehydrins have been identified in
sugarcane leaves with increased expression in response to heat
stress (Wahid and Close, 2007). Function of these proteins is
apparently related to protein degradation pathway, minimizing
the adverse effects of dehydration and oxidative stress during
heat stress (Schoffl et al., 1999).
In essence, expression of stress proteins is an important
adaptation toward heat-stress tolerance by plants. Of these,
expression of low and high molecular weight HSPs, widely
reported in a number of plant species, is the most important one.
These proteins show organelle- and tissue-specific expression
with deduced function like chaperones, folding and unfolding
of cellular proteins, and protection of functional sites from the
adverse effects of high temperature. Among other stress proteins,
expression of ubiquitin, Pir proteins, LEA and dehydrins has also

been established under heat stress. A main function of these


proteins appears to be protection of cellular and sub-cellular
structures against oxidative damage and dehydrative forces.
4. Mechanism of heat tolerance
Plants manifest different mechanisms for surviving under
elevated temperatures, including long-term evolutionary phenological and morphological adaptations and short-term avoidance
or acclimation mechanisms such as changing leaf orientation,
transpirational cooling, or alteration of membrane lipid compositions. In many crop plants, early maturation is closely
correlated with smaller yield losses under high temperatures,
which may be attributed to the engagement of an escape mechanism (Adams et al., 2001). Plants immobility limits the range
of their behavioral responses to environmental cues and places
a strong emphasis on cellular and physiological mechanisms of
adaptation and protection. Also, plants may experience different types of stress at different developmental stages and their
mechanisms of response to stress may vary in different tissues
(Queitsch et al., 2000). The initial stress signals (e.g., osmotic or
ionic effects, or changes in temperature or membrane fluidity)
would trigger downstream signaling processes and transcription
controls, which activate stress-responsive mechanisms to reestablish homeostasis and protect and repair damaged proteins
and membranes. Inadequate responses at one or more steps in the
signaling and gene activation processes might ultimately result
in irreversible damages in cellular homeostasis and destruction
of functional and structural proteins and membranes, leading
to cell death (Vinocur and Altman, 2005; Bohnert et al., 2006).
Even plants growing in their natural distribution range may experience high temperatures that would be lethal in the absence of
this rapid acclimation response. Furthermore, because plants can
experience major diurnal temperature fluctuations, the acquisition of thermotolerance may reflect a more general mechanism
that contributes to homeostasis of metabolism on a daily basis
(Hong et al., 2003). Mild stress episodes, however, should be
viewed as the acceleration of a program linked to the normal termination of phytomere production during the plant cycle, rather
than as an abrupt event linked to stress (Guilioni et al., 1997).
Elucidating the various mechanisms of plant response to
stress and their roles in acquired stress tolerance is of great
practical and basic importance. Some major tolerance mechanisms, including ion transporters, osmoprotectants, free-radical
scavengers, late embryogenesis abundant proteins and factors
involved in signaling cascades and transcriptional control are
essentially significant to counteract the stress effects (Wang et
al., 2004). Series of changes and mechanisms, beginning with
the perception of heat and signaling and production of metabolites that enable plants to cope with adversaries of heat stress,
have been proposed (Fig. 3). Heat stress effects are notable
at various levels, including plasma membrane and biochemical pathways operative in the cytosol or cytoplasmic organelles
(Sung et al., 2003). Initial effects of heat stress, however, are on
plasmalemma, which shows more fluidity of lipid bilayer under
stress. This leads to the induction of Ca2+ influx and cytoskeletal
reorganization, resulting in the upregulation of mitogen acti-

A. Wahid et al. / Environmental and Experimental Botany 61 (2007) 199223

211

Fig. 3. Proposed heat-stress tolerance mechanisms in plants. MAPK, mitogen activated protein kinases; ROS, reactive oxygen species; HAMK, heat shock activated
MAPK; HSE, heat shock element; HSPs, heat shock proteins; CDPK, calcium dependent protein kinase; HSK, histidine kinase. Partly adopted from Sung et al.
(2003).

vated protein kinases (MAPK) and calcium dependent protein


kinase (CDPK). Signaling of these cascades at nuclear level
leads to the production of antioxidants and compatible osmolytes
for cell water balance and osmotic adjustment. Production of
ROS in the organelles (e.g., chloroplast and mitochondria) is of
great significance for signaling as well as production of antioxidants (Bohnert et al., 2006). The antioxidant defense mechanism
is a part of heat-stress adaptation, and its strength is correlated with acquisition of thermotolerance (Maestri et al., 2002).
Accordingly, in a set of wheat genotypes, the capacity to acquire
thermotolerance was correlated with activities of CAT and SOD,

higher ascorbic acid content, and less oxidative damage (Sairam


and Tyagi, 2004).
One of the most closely studied mechanisms of thermotolerance is the induction of HSPs, which, as described in
above, comprise several evolutionarily conserved protein families. However, each major HSP family has a unique mechanism
of action with chaperonic activity. The protective effects of
HSPs can be attributed to the network of the chaperone machinery, in which many chaperones act in concert. An increasing
number of studies suggest that the HSPs/chaperones interact
with other stress-response mechanisms (Wang et al., 2004). The

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HSPs/chaperones can play a role in stress signal transduction


and gene activation (Nollen and Morimoto, 2002) as well as in
regulating cellular redox state (Arrigo, 1998). They also interact
with other stress-response mechanisms such as production of
osmolytes (Diamant et al., 2001) and antioxidants (Panchuk et
al., 2002).
Membrane lipid saturation is considered as an important element in high temperature tolerance. In a mutant wheat line
with increased heat resistance, heat treatment increased relative
quantities of linolenic acid (among galactolipids) and trans3-hexaldecanoic acid (among phospholipids), when compared
with the wild type (Behl et al., 1996). Currently, it is unknown
whether a higher or a lower degree of membrane lipid saturation
is beneficial for high-temperature tolerance (Klueva et al., 2001).
The contribution of lipid and protein components to membrane
function under heat stress needs further investigation. Localization of LMW-HSPs with chloroplastic membranes upon heat
stress suggests that they play a role in protecting photosynthetic
electron transport (Heckathorn et al., 1998).
An important component of thermotolerance is changes in
gene expression. Heat stress is known to swiftly alter patterns
of gene expression (Yang et al., 2006), inducing expression of
the HSP complements and inhibiting expression of many other
genes (Yost and Lindquist, 1986). The mRNAs encoding nonheat-stress-induced proteins are destabilized during heat stress.
Heat stress may also inhibit splicing of some mRNAs. Earlier
it was hypothesized that HSP-encoding mRNAs could not be
processed properly due to the absence of introns in the corresponding genes (Yost and Lindquist, 1986). Subsequently it was
shown that some HSP-encoding genes have introns and, under
heat-stress conditions, their mRNAs were correctly spliced
(Visioli et al., 1997). However, the mechanism of preferential
post-transcription modification and translation of HSP-encoding
mRNA under heat stress remains yet to be elucidated.
5. Acquired thermotolerance
Thermotolerance refers to the ability of an organism to cope
with excessively high temperatures. It has long been known that
plants, like other organisms, have the ability to acquire thermotolerance rather rapidly, may be within hours, so to survive under
otherwise lethal high temperatures (Vierling, 1991). The acquisition of thermotolerance is an autonomous cellular phenomenon
and normally results from prior exposure to a conditioning pretreatment, which can be a short but sublethal high temperature.
The acquisition of high level of thermotolerance protects cells
and organisms from a subsequent lethal heat stress. Thermotolerance can also be induced by a gradual increase in temperature
to lethal highs, as would be experienced under natural conditions
(Vierling, 1991), and induction in this way involves a number of
processes. Using Arabidopsis mutants, it was shown that, apart
from heat shock proteins (HSP32 and HSP101), ABA, ROS
and SA pathways are involved in the development and maintenance of acquired thermotolerance (Larkindale and Huang,
2005; Charng et al., 2006). Adaptive mechanisms that protect
cells from protoxic effects of heat stress are key factors in acquisition of thermotolerance. Examination of the adverse effects

caused by temperature extremes can reveal useful information, in


particular as heat-stress responses in plants are similar to those of
other stresses, including cold and drought (Rizhsky et al., 2002).
The heat shock response (HSR), defined as a transient reprogramming of gene expression, is a conserved biological reaction
of cells and organisms to elevated temperatures (Schoffl et al.,
1999). HSR has been of great interest for studying molecular mechanisms of stress tolerance and regulation of gene
expression in plants. The temperature for the induction of
HSR coincides with optimum growth temperature for any given
species, which is normally 510 C above normothermic conditions. The features of this response include induction of HSPs
and subsequently acquisition of a higher level of thermotolerance. The transient synthesis of HSPs suggests that the signal
triggering the response is either lost, inactivated or no longer recognized under conditions of long-term heat treatment (Schoffl,
2005). The involvement of HSPs in heat-stress tolerance is a
logical model, but direct support for function of HSPs in promoting thermotolerance has been difficult to obtain (Burke, 2001;
Schoffl, 2005).
In summary, thermotolerance acquired by plants via
autonomous synthesis of pertinent compounds or induced via
gradual exposure to heat episodes, though cost intensive, is an
important and potentially vital strategy. This phenomenon is
principally related to display of heat shock response and accomplished by reprogramming of gene expression, allowing plants
to cope with the heat stress.
6. Temperature sensing and signaling
Perception of stress and relay of the signal for turning on
adaptive response mechanisms are key steps towards plant stress
tolerance. There are multiple stress perceptions and signaling
pathways, some of which are specific while others may be
involved in cross-talk at various steps (Chinnusamy et al., 2004).
General responses to stress involve signaling of the stress via the
redox system. Chemical signals such as ROS, Ca2+ and plant
hormones activate genomic re-programing via signal cascades
(Joyce et al., 2003; Suzuki and Mittler, 2006). Although the
presence of a plant thermometer has not been established, it
is suggested that changing membrane fluidity plays a central
role in sensing and influencing gene expression both under high
and low temperatures. This suggests that sensors are located in
microdomains of membranes, which are capable of detecting
physical phase transition, eventually leading to conformational
changes and/or phosphorylation/dephosphorylation cycles due
to changes in temperature (Plieth, 1999). In this regard, a model
for temperature sensing and regulation of heat shock response
integrates observed membrane alterations. Changes in the ratio
of saturated to unsaturated fatty acid on the set point of temperature for the heat shock response (HSR) alters activities of
HSFs.
Rigidification of thylakoid membranes appears to invoke
altered expression profiles of heat shock genes, suggesting that
the temperature sensing mechanism may be located on the thylakoid membrane (Horvath et al., 1998). The prospect of the
thylakoid membrane acting as a heat sensor is physiologically

A. Wahid et al. / Environmental and Experimental Botany 61 (2007) 199223

crucial, because it is susceptible to temperature upshift, owing


to its highly unsaturated character, and the presence of photosystems, which are fragile to temperature changes (Sung et al.,
2003).
Various signaling ions and molecules are involved in temperature sensing and signaling. As a signaling response to
temperature stress, cytosolic Ca2+ sharply rises (Larkindale and
Knight, 2002), which seems to be linked to the acquisition
of tolerance possibly by transducing high temperature-induced
signals to MAPK. MAPK cascades are important parts of signal transduction pathways in plants and thought to function
ubiquitously in many responses to external signals (Kaur and
Gupta, 2005). A heat-shock activated MAPK (HAMK) has been
identified, the activation of which was triggered by apparent
opposite changes in membrane fluidity coupled with cytoskeletal remodeling (Sangwan and Dhindsa, 2002). Ca2+ influx and
the action of Ca-dependent protein kinases (CDPK) have been
closely correlated with the expression of HSPs (Sangwan and
Dhindsa, 2002). However, another study suggested that Ca2+
is not required for production of HSPs in plants, despite the
fact that heat stress induces uptake of Ca2+ and induction of
some calmodulin (CaM) related genes (Gong et al., 1997). As
a mediator of Ca2+ signal, CaM is activated by binding Ca2+ ,
inducing a cascade of regulatory events and regulation of many
HSP genes (Liu et al., 2003). Several studies have shown that
Ca2+ is involved in the regulation of plant responses to various
environmental stresses, including high temperature. Increasing
cytosolic Ca2+ content under heat stress may alleviate heat
injury, such as increased activity of antioxidants (Gong et al.,
1997), turgor maintenance in the guard cells (Webb et al., 1996)
and enable plant cells to better survive. However, excessive
Ca2+ released into the cytosol and sustained high cytosolic Ca2+
concentration might be cytotoxic (Wang and Li, 1999).
Specific groups of potential signaling molecules like SA,
ABA, CaCl2 , H2 O2 , and ACC may induce tolerance of plants
to heat stress by reducing oxidative damage (Larkindale and
Huang, 2004). Being molecules of somewhat novel interest in
the last few years, H2 O2 and NO have emerged to be central players in the world of plant cell signaling under stressful situations
(Dat et al., 2000). A protein phosphorylation cascade has been
shown to be activated by H2 O2 is a MAPK cascade. Methyl-SA
has a major signaling role in the gene activation under heat stress
up to 1.8 nmol g1 dry mass of tissue, beyond which it becomes
lethal to cell metabolism (Llusia et al., 2005).
In short, sensing of high temperature and induction of signaling cascades are important adaptive steps in coping with
adversaries of heat stress. Although numerous molecules including ROS, hormones and ethylene have been identified for the
perception of heat stress cues, role of Ca2+ is exclusive.
7. Genetic improvement for heat-stress tolerance
Recent studies have suggested that plants experience oxidative stresses during the initial period of adjustment to any
stress. Plant responses to stress progress from general to specific. Specific responses require sustained expression of genes
involved in processes specific to individual stresses (Yang et

213

al., 2006). These responses accommodate short-term reaction


or tolerance to specific stresses. However, genome plasticity in plants, including genetic (e.g., directed mutation) and
epigenetic (e.g., methylation, chromatin remodeling, histone
acetylation) changes, allows long-term adaptation to environmental changes/conditions (Joyce et al., 2003). Such adaptations
may be necessary for long-term survival or establishment of
plant genotypes/species in particular environmental niches.
Under agricultural systems, plants adaptation or their tolerance to environmental stresses can be manipulated by various
approaches.
In general, the negative impacts of abiotic stresses on agricultural productivity can be reduced by a combination of
genetic improvement and cultural practices. Genetic improvement entails development of cultivars which can tolerate
environmental stresses and produce economic yield. Adjustment/modifications in cultural practices, such as planting time,
plant density, and soil and irrigation managements, however, can
minimize stress effects, for example by synchronizing the stresssensitive stage of the plant with the most favorable time period of
the season. In practice, to be successful in improving agricultural
productivity in stress environments, both genetic improvement
and adjustment in cultural practices must be employed simultaneously. Agriculturists have long been aware of desirable
cultural practices to minimize adverse effects of environmental stresses on crop production. However, genetic improvement
of crops for stress tolerance is relatively a new endeavor and has
been considered only during the past 23 decades. Traditionally,
most plant breeding programs have focused on development
of cultivars with high yield potential in favorable (i.e., nonstress) environments. Such efforts have been very successful
in improving the efficiency of crop production per unit area
and have resulted in significant increases in total agricultural
production (Warren, 1998). However, genetic improvement of
plants for stress tolerance can be an economically viable solution for crop production under stressful environments (Blum,
1988). The progress in breeding for stress tolerance depends
upon an understanding of the physiological mechanisms and
genetic bases of stress tolerance at the whole plant, cellular and
molecular levels. Considerable information is presently available regarding the physiological and metabolic aspects of plant
heat-stress tolerance, as discussed earlier. However, information regarding the genetic basis of heat tolerance is generally
scarce, though the use of traditional plant breeding protocols
and contemporary molecular biological techniques, including
molecular marker technology and genetic transformation, have
resulted in genetic characterization and/or development of plants
with improved heat tolerance. In particular, the application of
quantitative trait locus (QTL) mapping has contributed to a better understanding of the genetic relationship among tolerances
to different stresses. In below, a summary of such efforts and
progresses is presented and discussed.
7.1. Conventional breeding strategies
Physiological and genetic investigations indicate that most
abiotic stress tolerance traits are complex, controlled by more

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than one gene, and highly influenced by environmental variation (Blum, 1988). The quantification of tolerance often poses
serious difficulties. Direct selection under field conditions is
generally difficult because uncontrollable environmental factors
adversely affect the precision and repeatability of such trials.
Often, no consistent high-temperature conditions can be guaranteed in field nurseries, as heat stress may or may not occur in the
field. Furthermore, stress tolerance is a developmentally regulated, stage-specific phenomenon; tolerance at one stage of plant
development may not be correlated with tolerance at other developmental stages. Individual stages throughout the ontogeny of
the plant should be evaluated separately for the assessment of
tolerance and for the identification, characterization and genetic
manipulation of tolerance components. Moreover, species may
show different sensitivity to heat stress at different developmental stages. For example, in tomato, though plants are sensitive
to high temperatures throughout the plant ontogeny, flowering
and fruit set are the most sensitive stages; fruit set is somewhat affected at day/night temperatures above 26/20 C and
is severely affected above 35/26 C (Berry and Rafique-Uddin,
1988). Thus, partitioning of the tolerance into its developmental and physiological/genetic components may provide a better
understanding of the plants response to heat stress and facilitate development of plants with stress tolerance throughout the
plants life cycle.
A common method of selecting plants for heat-stress tolerance has been to grow breeding materials in a hot target
production environment and identify individuals/lines with
greater yield potential (Ehlers and Hall, 1998). Under such
conditions, however, the presence of other stresses such as
insect-pests has made the selection process very difficult, particularly during reproductive stage. A suggested approach has been
to identify selection criteria during early stages of plant development, which may be correlated with heat tolerance during
reproductive stages. Unfortunately, thus far no reliable criteria
have been identified. A rather more effective approach has been
development of glasshouses for heat tolerance screening. Theoretically, such nurseries can be utilized for screening throughout
the plant life cycle, from seedling to reproductive stages. An
advantage of glasshouse screening is that the required temperature conditions can be maintained consistently for the duration of
the experiment. Also, because a key factor in screening for heat
tolerance is maintaining high night temperatures, glasshouse
nursery can provide such conditions more reliably than field
nurseries. However, in many places in the world where high
temperatures are a concern, such growth/glasshouse facilities
are nonexistent or limited in size, precluding screening of large
breeding populations.
A major challenge in traditional breeding for heat tolerance is the identification of reliable screening methods and
effective selection criteria to facilitate detection of heat-tolerant
plants. Several screening methods and selection criteria have
been developed/proposed by different researchers. For example, a heat tolerance index (HTI) for growth recovery after heat
exposure was proposed for sorghum (Young et al., 2001). The
index is the ratio of the increase in coleoptile length after finite
exposure to heat stress (e.g., 50 C) to the increase in coleoptile

length in the no-stress treatment. This approach allows a rapid


and repeated recording of coleoptile length, which may be used
to screen a large number of genotypes in a rather short period
of time. Although this is a very cost effective and easy-to-assay
technique of screening for heat tolerance, its correlation with
performance under field conditions and its effectiveness in different crop species are yet unknown (Setimela et al., 2005). In
some crop species such as tomato, a strong positive correlation
has been observed between fruit set and yield under high temperature. Thus, evaluation of germplasm to identify sources of
heat tolerance has regularly been accomplished by screening
for fruit set under high temperature (Berry and Rafique-Uddin,
1988). Furthermore, although poor fruit set at high temperature
cannot be attributed to a single factor, decreases in pollen germination and/or pollen tube growth are among the most commonly
reported factors. Therefore, pollen viability has been suggested
as an additional indirect selection criterion for heat tolerance.
Also, production of viable seed is often reduced under heat stress
and thus high seed set has been arguably reported as an indication
of heat tolerance (Berry and Rafique-Uddin, 1988).
Because pollen viability and fertility is adversely affected
by high temperature, any type of fruit and seed production that
may not need sexual hybridization and fertilization may provide
heat tolerance. For example, apomixes, in particular the types
that assure reproduction without the need for pollination may
be very useful when developing cultivars for production under
high temperatures. Through genetic engineering it may be possible to insert the cassette of genes needed to confer facultative
apomixis. Currently, considerable research is underway to identify genes or enzymes that may be involved in production of
apomixis (Albertini et al., 2005). Among many other traits which
are affected by high temperature, the non-reproductive processes
include photosynthetic efficiency, assimilate translocation, mesophyll resistance and disorganization of cellular membranes
(Chen et al., 1982). Breeding to improve such traits under high
temperatures may result in the development of cultivars with
heat tolerance attributes.
Several other issues of concern when employing traditional
breeding protocols to develop heat-tolerant crop plants are as
follows:
1. Identification of genetic resources with heat tolerance
attributes. In many plant species, for example soybeans and
tomatoes, limited genetic variations exist within the cultivated species necessitating identification and use of wild
accessions. However, often there are great difficulties in both
the identification and successful use of wild accessions for
stress tolerance breeding (Foolad, 2005).
2. When screening different genotypes (in particular wild accessions) for growth under high temperatures, distinction must
be made between heat tolerance and growth potential. Often
plants with higher growth potential perform better regardless
of the growing conditions.
3. When breeding for stress tolerance, often it is necessary
that the derived lines/cultivars be able to perform well
under both stress and non-stress conditions. Development of
such genotypes is not without inherent difficulties. In some

A. Wahid et al. / Environmental and Experimental Botany 61 (2007) 199223

plant species, heat tolerance is often associated with some


undesirable horticultural or agronomical characteristics. In
tomato, for example, two undesirable characteristics commonly observed in heat-tolerant genotypes are small fruit
and restricted foliar canopy (Scott et al., 1997). The production of small fruit is most likely due to adverse effects of high
temperature on the production of auxins in the fruit, and the
poor canopy is due to the highly reproductive nature of the
heat-tolerant genotypes.
In summary, breeding for heat tolerance is still in its infancy
stage and warrants more attention than it has been given in the
past. It is unfortunate that the literature contains relatively little information on breeding for heat tolerance in different crop
species. However, despite all the complexity of heat tolerance
and difficulties encountered during transfer of tolerance, some
heat-tolerant inbred lines and hybrid cultivars with commercial
acceptability have been developed and released, at least in a
few crop species such as tomato (Scott et al., 1986; Scott et al.,
1995). Nevertheless, to accelerate such progresses, major areas
of emphasis in the future should be: (1) designing/development
of accurate screening procedures; (2) identification and characterization of genetic resources with heat tolerance; (3) discerning
the genetic basis of heat tolerance at each stage of plant development; (4) development and screening of large breeding populations to facilitate transfer of genes for heat tolerance to commercial cultivars (Siddique et al., 1999). The use of advanced
molecular biology techniques may facilitate development of
plants with improved heat tolerance, as described in below.
7.2. Molecular and biotechnological strategies
Recent genetic studies and efforts to understand/improve
high-temperature tolerance of crop plants using traditional protocols and transgenic approaches have largely determined that
plant heat-stress tolerance is a multigenic trait. Different components of tolerance, controlled by different sets of genes, are
critical for heat tolerance at different stages of plant development
or in different tissues (Howarth, 2005; Bohnert et al., 2006).
Thus, the use of genetic stocks with different degrees of heat
tolerance, correlation and co-segregation analyses, molecular
biology techniques and molecular markers to identify tolerance
QTLs are promising approaches to dissect the genetic basis of
thermotolerance (Maestri et al., 2002). Most recently, biotechnology has contributed significantly to a better understanding of
the genetic basis of heat tolerance. For example, several genes
responsible for inducing the synthesis of HSPs have been identified and isolated in various plant species, including tomato and
maize (Liu et al., 2006; Sun et al., 2006; Momcilovic and Ristic,
2007). Also, it has been determined that induction of many
heat-inducible genes is attributed to the conserved heat shock
elements (HSEs), which are located in the TATA box proximal
5 flanking regions of heat shock genes (Schoffl et al., 1999). The
requirement of TATA box was earlier demonstrated by deletion
analysis of soybean heat shock genes in sunflower (Czarnecka
et al., 1989). In addition, a number of other sequence motifs
have been identified in plants that have quantitative effects on

215

expression of certain heat shock genes. For example, there is evidence for the involvement of CCAAT box and AT-rich sequences
(Czarnecka et al., 1989). The HSEs are the binding sites for the
transitive heat shock transcription factor (HSF), the activation of
which in higher eukaryotes is a multi-step process. In response
to heat stress, HSF is converted from a monomeric to trimeric
form. The trimeric HSF is localized predominantly in the nucleus
and has a high affinity of binding to HSEs. It is believed that
interaction of HSF with HSP70 or other HSPs results in the activation of HSF via conformational changes involving monomer to
trimer transition and nuclear targeting (Schoffl et al., 1999). This
expression of heat shock genes is modulated by the temperature
within permissive range, thereby conferring plant thermotolerance (Yang et al., 2006). Further research has demonstrated that
thermotolerance of plants also can be modulated/effected by
changes in transcriptional and translational activities. Ongoing
transcription is needed during stress to support a basal level
of translational activity in the subsequent recovery from the
stress, but it does not appear to be required for the heat-mediated
increase in mRNA stability (Gallie and Pitto, 1996). In general,
such activities in plants undergo rapid changes during developmental stages such as seed formation and germination, and
also during abiotic stresses such as heat shock, hypoxia and
wounding (Gallie et al., 1998).
Two common biotechnological approaches to study and
improve plant stress tolerance include marker-assisted selection (MAS) and genetic transformation. During the past two
decades, the use of these approaches has contributed greatly to
a better understanding of the genetic and biochemical bases of
plant stress-tolerance and, in some cases, led to the development
of plants with enhanced tolerance to abiotic stress. Because of
the general complexity of abiotic stress tolerance and the difficulty in phenotypic selection for tolerance, MAS has been
considered as an effective approach to improve plant stress tolerance (Foolad, 2005). The use of this approach, however, requires
identification of genetic markers that are associated with genes
or QTLs affecting whole plant stress tolerance or individual
components contributing to it. During the past two decades,
substantial amounts of research has been conducted in different
crop species to identify genetic markers associated with different environmental stresses, in particular drought, salinity and
low temperatures. For example, molecular marker technology
has allowed the identification and genetic characterization of
QTLs with significant effects on stress tolerance during different stages of plant development and facilitated determination
of genetic relationships among tolerance to different stresses
(Foolad, 2005). Comparatively, however, limited research has
been conducted to identify genetic markers associated with heat
tolerance in different plant species. In Arabidopsis, for example,
four genomic loci (QTLs) determining its capacity to acquire
thermotolerance were identified using a panel of heat-sensitive
mutants (Hong and Vierling, 2000). Use of restriction fragment
length polymorphism (RFLP) revealed mapping of eleven QTLs
for pollen germination and pollen tube growth under heat stress
in maize (Frova and Sari-Gorla, 1994).
Recent advances in genetic transformation and gene
expression techniques have contributed greatly to a better

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understanding of the genetic and biochemical bases of plant


stress-tolerance and, in some cases, led to the development of
plants with enhanced tolerance to abiotic stresses. For example, a significant progress has been made in the identification
of genes, enzymes or compounds with remarkable effects
on plant stress tolerance at the cellular or organismal level
(Apse and Blumwald, 2002; Bohnert et al., 2006). Furthermore,
manipulation of the expression or production of the identified
genes, proteins, enzymes, or compounds through transgenic
approaches have resulted in the development of plants with
enhanced stress tolerance in different plant species (Zhang et
al., 2001; Rontein et al., 2002). However, a major limitation in
the use of such techniques for improving plant high-temperature
tolerance is that the critical factors conferring the enhanced temperature tolerance in higher plants are still poorly understood.
Initial research on molecular manipulation to improve plant
heat tolerance focused on production of enzymes that detoxify reactive oxygen species, including SOD. Reactive oxygen
species are induced by most types of stresses (Havaux, 1998;
Sairam and Tyagi, 2004) and their production has been envisaged in stress cross-tolerance (Allan et al., 2006). In addition to
increased production of SOD, many other potential approaches
can be utilized to detoxify ROS and produce plants tolerant
of heat stress (Zidenga, 2005). If the critical components are
determined, genetic engineering technology can be utilized to
incorporate thermo-tolerance into adapted cultivars. Despite
the very many limitations, some progress has been made. For
example, transgenic tobacco plants with altered chloroplast
membranes by silencing the gene encoding chloroplast omega3 fatty acid desaturase have been produced which produce
less trienoic fatty acids and more dienoic fatty acids in their
chloroplasts than the wild type. These plants exhibited greater
photosynthesis and grew better than wild type plants under high
temperatures (Murakami et al., 2000). Dnak1, a gene responsible for high salt tolerance in the cyanobacterium Aphanothece
halophytica, when transferred into tobacco was expressed and
conferred high temperature resistance (Ono et al., 2001). Development of plants capable of higher production of glycinebetaine
through transformation with the BADH gene has been suggested
as a potentially effective method to enhance heat tolerance in
plants (Yang et al., 2005). Thermal stability of rubisco activase,
a molecular chaperone responsible for the activity of rubisco,
is important in maintaining its activity (Salvucci and CraftsBrandner, 2004a). By transforming tobacco plants with rubisco
activase gene, thermotolerance is achieved by reversible decarboxylation of rubiscoa likely protective mechanism by which
the plant protects its photosynthetic apparatus (Sharkey et al.,
2001).
Genetic engineering of heat shock factors (HSF) and antisense strategies are instrumental to the understanding of both the
functional roles of HSPs and the regulation of HSFs. Manipulations of the HS-response in transgenic plants have the potential
to improve common abiotic stress tolerance and this may have
a significant impact on the exploitation of the inherent genetic
potential of agronomically important plants. Transgenic overexpression of certain HSFs and HSF-fusion proteins results
in an expression of HSPs at normal temperature (Iba, 2002).

The increased acquired thermotolerance of transgenic lines is


attributed to the higher levels of HSP chaperones. It has also
been demonstrated that tomato MT-sHSP has a molecular chaperone function in vitro (Liu and Shono, 1999) and recently it
has been demonstrated that MT-sHSP gene exhibits thermotolerance in transformed tobacco with the tomato MT-sHSP
gene (Sanmiya et al., 2004) at the plant level. Experimental
data obtained from transgenic, reverse-genetics and mutation
approaches in non-cereal species confirm causal involvement
of HSPs in thermotolerance in plants (Queitsch et al., 2000).
However, the cellular targets of HSPs are still unknown.
In summary, transformation technology for improving plant
stress tolerance is still at its infancy, and the success to date represents only a beginning. Advancements in marker technology and
genetic transformation are expected to contribute significantly
to the development of plants with tolerance to high temperatures in future. With the current transformation technology, it
is becoming possible to transfer multiple genes, which may act
synergistically and additively to improve plant stress tolerance.
Future knowledge of tolerance components and the identification and cloning of responsible genes may allow transformation
of plants with multiple genes and production of highly stresstolerant transgenic plants. In addition, there is no report to date
of any studies testing the performance of transgenic plants under
field stress conditions. Therefore, much more work is needed to
gain a clearer understanding of the genetics, biochemical and
physiological basis of plant heat tolerance.
8. Induction of heat tolerance
Although genetic approaches may be beneficial in the production of heat-tolerant plants, it is likely that the newly produced
plants are low yielding compared to near-isogenic heat sensitive plants. Thus, considerable attention has been devoted
to the induction of heat tolerance in existing high-yielding
cultivars. Among the various methods to achieve this goal,
foliar application of, or pre-sowing seed treatment with, low
concentrations of inorganic salts, osmoprotectants, signaling
molecules (e.g., growth hormones) and oxidants (e.g., H2 O2 )
as well as preconditioning of plants are common approaches.
High-temperature preconditioning has been shown to drastically
reduce the heat-induced damage to black spruce seedlings at
moderately high temperatures (Colclough et al., 1990). Preconditioned tomato plants exhibited good osmotic adjustment by
maintaining the osmotic potential and stomatal conductance,
and better growth than non-conditioned plants (Morales et al.,
2003). Similarly, heat acclimated, compared to non-acclimated,
turfgrass leaves manifested higher thermostability, lower lipid
peroxidation product malondialdehyde (MDA) and lower damage to chloroplast upon exposure to heat stress (Xu et al., 2006).
In pearl millet, pre-sowing hardening of the seed at high
temperature (42 C) resulted in plants tolerant to overheating
and dehydration and showing higher levels of water-soluble
proteins and lower amounts of amide-N in leaves compared
to non-hardened plants (Tikhomirova, 1985). The researchers
proposed that the higher heat tolerance was due to enhanced
glutathione synthase activity, promoting binding of the ammo-

A. Wahid et al. / Environmental and Experimental Botany 61 (2007) 199223

nia accumulated during exposure to high temperature. In tomato,


it was demonstrated that heat treatment administered to plants
prior to chilling stress resulted in reduced incidence and severity
of chilling injury in fruit and other organs (Whitaker, 1994). In
some cool season grasses, under heat stress, Ca2+ is required
for maintenance of antioxidant activity and not for osmotic
adjustment (Jiang and Haung, 2001). Under heat stress, Ca2+
requirement for growth is high to mitigate adverse effects of
the stress (Kleinhenz and Palta, 2002). It has been shown that
exogenous application of Ca2+ promotes plants heat tolerance.
Application of Ca2+ in the form of CaCl2 prior to the stress treatment elevated the content of lipid peroxidation product, MDA
and stimulated the activities of guaiacol peroxidase, SOD and
catalase, which could be the reasons for the induction of heat
tolerance (Kolupaev et al., 2005).
Among the low molecular weight organic compounds,
glycinebetaine and polyamines have been successfully applied
to induce heat tolerance in various plant species. For example,
barley seeds pre-treated with glycinebetaine led to plants with
lower membrane damage, better photosynthetic rate, improved
leaf water potential and greater shoot dry mass, compared
to untreated seeds (Wahid and Shabbir, 2005). Also, exogenous application of 4 mM spermidine improved tomato heat
resistance by improving chlorophyll fluorescence properties,
hardening and higher resistance to thermal damage of the
pigment-protein complexes structure, and the activity of PSII
during linear increase in temperature (Murkowski, 2001). Thus,
to improve plant heat tolerance, alternative approaches to genetic
means would include pre-treatment of plants or seeds with heat
stress or certain mineral or organaic compounds. The success of
such approach, however, depends on plant species and gentoypes
and must be studied on case basis.
9. Energy economics under heat stress
Reduction in plant growth is a major consequence of growing
under stress conditions. This occurs mainly due to a reduction
in net photosynthesis rate and generation of reducing powers as
well as interference with mitochondrial functions. It is suggested
that during light reactions increased leaf temperature induces
ATP synthesis to balance ATP consumption under heat stress
possibly by cyclic electron flow (Bukhov et al., 1999). During
dark reactions of photosynthesis, rubisco activation in Calvin
cycle has been determined as a critical step, being inhibited at
3540 C, which results in decreased net CO2 assimilation and
the production of carbohydrates (Crafts-Brandner and Salvucci,
2000; Dubey, 2005). In mitochondria, environmental stress normally causes NAD+ breakdown, ATP over-consumption and
higher rate of respiration. This is partially due to a breakdown in
the NAD+ pool caused by the enhanced activity of poly(ADPribose) polymerase (PARP), which uses NAD+ as a substrate
to synthesize polymers of ADP-ribose. This poly(ADP) ribosylation is a post-translational modification of nuclear proteins
that seems to be initiated by oxidative and other types of DNA
damage. Stress-induced depletion of NAD+ results in a similar
depletion of energy, since ATP molecules are required to resynthesize the depleted NAD+ (Zidenga, 2005). Collectively,

217

these reactions deplete the energy of the plant and enhance


the production of ROS, which eventually lead to cell death
(De Block et al., 2005). A strategy of improving stress tolerance in plants by maintaining the plants energy homeostasis
under stress is the production of transgenic plants with lowered poly(ADP) ribosylation activity; such transgenics appear
to be tolerant to multiple stresses by preventing energy overconsumption under stress, thereby allowing normal mitochondrial
respiration (De Block et al., 2005). In short, heat tolerance in
plants is a cost-intensive process and consumes considerable
cellular energy to cope with adversaries of high temperature.
10. Conclusion and future prospects
Plants exhibit a variety of responses to high temperatures,
which are depicted by symptomatic and quantitative changes in
growth and morphology. The ability of the plant to cope with
or adjust to the heat stress varies across and within species as
well as at different developmental stages. Although high temperatures affect plant growth at all developmental stages, later
phenological stages, in particular anthesis and grain filling, are
generally more susceptible. Pollen viability, patterns of assimilate partitioning, and growth and development of seed/grain
are highly adversely affected. Other notable heat stress effects
include structural changes in tissues and cell organelles, disorganization of cell membranes, disturbance of leaf water relations,
and impedance of photosynthesis via effects on photochemical and biochemical reactions and photosynthetic membranes.
Lipid peroxidation via the production of ROS and changes in
antioxidant enzymes and altered pattern of synthesis of primary
and secondary metabolites are also of considerable importance.
In response to heat stress, plants manifest numerous adaptive changes. The induction of signaling cascades leading to
profound changes in specific gene expression is considered an
important heat-stress adaptation. Although various signaling
molecules are synthesized under heat stress, the role of Ca2+
remains critical. A fundamental heat-stress response ubiquitous to plants is the expression of HSPs, which range from
low (10 kDa) to high (100 kDa) molecular mass in different
species. Evidence on synthesis and accumulation of some other
stress-related proteins is also available. Such stress proteins are
thought to function as molecular chaperones, helping in folding
and unfolding of essential proteins under stress, and ensuring
three-dimensional structure of membrane proteins for sustained
cellular functions and survival under heat stress.
In addition to genetic means to developing plants with
improved heat tolerance, attempts have been made to induce heat
tolerance in a range of plant species using different approaches.
These include preconditioning of plants to heat stress and exogenous applications of osmoprotectants or plant growth-regulating
compounds on seeds or whole plants. Results from such applications are promising and further research is forthcoming. Also,
while some notable progress has been reported as to the development of crop plants with improved heat tolerance via traditional
breeding, the prospect for engineering plants with heat tolerance
is also good considering accumulating molecular information on
the mechanisms of tolerance and contributing factors.

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Although physiological mechanisms of heat tolerance are relatively well understood, further studies are essential to determine
physiological basis of assimilate partitioning from source to
sink, plant phenotypic flexibility which leads to heat tolerance,
and factors that modulate plant heat-stress response. Furthermore, applications of genomics, proteomics and trascriptomics
approaches to a better understanding of the molecular basis of
plant response to heat stress as well as plant heat tolerance are
imperative. As in the case of most other abiotic stresses, foliar
plant parts are more directly impinged upon by high temperatures than roots. However, an understanding of root responses
to heat stress, most likely involving rootshoot signaling, is crucial and warrants further exploration. Molecular knowledge of
response and tolerance mechanisms will pave the way for engineering plants that can tolerate heat stress and could be the basis
for production of crops which can produce economic yield under
heat-stress conditions.
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